- Email: [email protected]
EP4-cAMP-Epac-Dependent Pathway
Abstracts AB265
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
Endogenous PGE2 Amplifies IL-33 Production By Macrophages through an EP2/EP4-cAMP-EpacDependent Pathway
Sachin K. Samuchiwal, PhD1,2, Barbara Balestrieri, MD1,2, Amanda Paskavitz1, Hannah Raff1, and Joshua A. Boyce, MD, FAAAAI1,2; 1Brigham and Women’s Hospital, Division of Rheumatology, Immunology, and Allergy, Boston, MA, 2Harvard Medical School, Department of Medicine, Boston, MA. RATIONALE: Macrophages activated through toll-like receptors can generate IL-33, an IL-1 family cytokine that induces innate immune responses through its receptor, ST2. Lipopolysaccharide (LPS) induces macrophages to generate prostaglandin-E2 (PGE2), a cyclooxygenase (COX)-2 and dominantly microsomal PGE2 synthase-1 (mPGES-1) product. The effects of PGE2 on IL-33 production by macrophages are not known. METHODS: We studied IL-33 production by bone-marrow derived murine macrophages (bmMF) by LPS in absence of endogenous PGE2 and in presence of exogenous PGE2. We elucidated underlying mechanisms using pharmacological approaches in bmMFs from mice respectively lacking mPGES-1 and EP2receptors. RESULTS: IL-33 production by bmMFs requires endogenous PGE2 and intrinsic expression of EP2 receptor to amplify NF-kB-dependent LPSinduced IL-33 expression via exchange protein activated by cAMP (EPAC). Selective agonists of EP2 and to a lesser extent EP4, but not of EP1 or EP3, potentiated LPS-induced IL-33 generation from mPGES-1null and WT bmMFs. Exogenous PGE2 enhanced IL-33 mRNA expression via a cAMP dependent pathway and its effect was also mimicked by an EPAC-selective agonist, but not by a PKA-selective agonist, and was attenuated by an EPAC-selective antagonist or knockdown. Although both p38 MAPK and NF-kB activations were needed for IL-33 production, they occurred independently of PGE2. CONCLUSIONS: Our data demonstrate that endogenous PGE2 is required to amplify LPS-induced IL-33 expression in mouse bmMFs, involving cAMP dependent pathway involving EPAC. The ubiquitous induction of mPGES-1-dependent PGE2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders.
833
Environmental Adjuvants Induce Neuropilin-2 Expression in Human and Murine Alveolar Macrophages
Timothy P. Moran, MD, PhD1, Robert M. Immormino, PhD2, Hideki Nakano, PhD3, Neil Alexis, PhD4, Andrew J. Ghio, MD5, David B. Peden, MD, MS, FAAAAI6, and Donald N. Cook, PhD7; 1University of North Carolina School of Medicine, Chapel Hill, NC, 2Center for Environmental Medicine, Asthma and Lung Biology, Chapel Hill, NC, 3National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 4Center Environmental Med. Asthma/Lung Biology, Chapel Hill, NC, 5US EPA, Chapel Hill, NC, 6University of North Carolina at Chapel Hill School Medicine, Chapel Hill, NC, 7National Institute of Environmental Health Sciences, Research Triangle Park, NC. RATIONALE: Neuropilin-1 (NRP1) and -2 (NRP2) are transmembrane receptors expressed by lung macrophages, but their functional importance is unclear. Here, we investigated the effect of environmental adjuvants on NRP1 and NRP2 expression in human and murine alveolar macrophages (AM). METHODS: Human AM or murine bone marrow-derived macrophages (BMM) were treated ex vivo with Toll-like receptor (TLR) ligands, house dust extract (HDE) or proteases, and NRP1 and NRP2 expression was evaluated by real-time quantitative PCR and confocal microscopy. NRP2 reporter mice were also exposed to HDE or endotoxin (TLR4 agonist) in vivo, and NRP2 expression by AM was determined by flow cytometry. RESULTS: Ex vivo stimulation of human AM or murine BMM with endotoxin resulted in increased NRP2 but decreased NRP1 expression. NRP2 was also induced in BMM treated with HDE and agonists for TLR2,
TLR3 and TLR9. In contrast, protease treatment of BMM did not induce significant NRP2 expression. Expression of the NRP2 co-receptors, plexinA3 and vascular endothelial growth factor (VEGF) receptor 3, was increased in endotoxin- and HDE-stimulated BMM. In vivo airway exposure to HDE or endotoxin induced NRP2 expression in murine AM. Furthermore, quantitative PCR analysis revealed expression of NRP2 ligands (VEGF and semaphorin 3F) in lungs of HDE-exposed mice. CONCLUSIONS: Stimulation of innate immune receptors by TLR ligands and HDE induces NRP2 expression in human and murine AM. Given the importance of AM in maintaining immune homeostasis in the lungs, our findings suggest that NRP2 may play a role in regulating airway inflammation in response to environmental adjuvants.
834
Oxidative Burst Defines Differences in Respiratory Virus Associated Neutrophil Subtypes
Michelle Rohlfing1, Dorothy S. Cheung, MD, FAAAAI2, and Mitchell H. Grayson, MD, FAAAAI3; 1Nationwide Children’s Hospital, Columbus, OH, 2Genentech, South San Francisco, CA, 3700Children’s Drive, Nationwide Children’s Hospital / The Ohio State University, Columbus, OH. RATIONALE: Neutrophil (PMN) subsets are important in atopic disease development. In the Sendai virus (SeV) model of post-viral atopic disease, we demonstrated a critical need for CD49d+ PMN driving disease development. We showed humans express CD49d+ PMN and they track with atopy. However, unlike SeV, inoculation of mice with lipopolysaccharide (LPS) recruits CD49d– PMNs. Differences between CD49 expressing and nonexpressing PMNs are not well known. We undertook this study to determine if CD49d expression was associated with altered PMN oxidative burst. METHODS: C57BL/6 mice were inoculated intranasally with 2x105 pfu SeV. On day 3 post-inoculation, flow sorting obtained CD49d+ and CD49d– BAL PMN based on Gr-1/CD49d expression. Respiratory burst was examined using DHR assay (Cayman), with unstained subsets as controls. Additional PMN samples were cultured with 200 nM PMA for 45 min before DHR assay. BAL PMN (all CD49d–) from mice inoculated intranasally with 10 mg LPS one-day prior also were examined. RESULTS: SeV increased respiratory burst in CD49d+ compared to _4, CD49d– PMN (296630 Net MFI versus 114664, mean6sem, n> p50.03). PMA treatment had no effect (2856102 versus 7263, p50.05). However, LPS CD49d– PMN demonstrated significantly greater burst (312617 and 353625 PMA) than SeV CD49d– PMN (p50.03 for _0.3 for both). both), but not CD49d+ PMN (p> CONCLUSIONS: In SeV infection, CD49d+ have greater oxidative burst than CD49d– PMN; however, CD49d– PMN have an impaired burst compared to LPS CD49d– PMN. These data demonstrate diversity in PMN subtypes; further research will clarify the ‘‘PMN to atopy’’ axis.
MONDAY
832
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
Endogenous PGE2 Amplifies IL-33 Production By Macrophages through an EP2/EP4-cAMP-EpacDependent Pathway
Sachin K. Samuchiwal, PhD1,2, Barbara Balestrieri, MD1,2, Amanda Paskavitz1, Hannah Raff1, and Joshua A. Boyce, MD, FAAAAI1,2; 1Brigham and Women’s Hospital, Division of Rheumatology, Immunology, and Allergy, Boston, MA, 2Harvard Medical School, Department of Medicine, Boston, MA. RATIONALE: Macrophages activated through toll-like receptors can generate IL-33, an IL-1 family cytokine that induces innate immune responses through its receptor, ST2. Lipopolysaccharide (LPS) induces macrophages to generate prostaglandin-E2 (PGE2), a cyclooxygenase (COX)-2 and dominantly microsomal PGE2 synthase-1 (mPGES-1) product. The effects of PGE2 on IL-33 production by macrophages are not known. METHODS: We studied IL-33 production by bone-marrow derived murine macrophages (bmMF) by LPS in absence of endogenous PGE2 and in presence of exogenous PGE2. We elucidated underlying mechanisms using pharmacological approaches in bmMFs from mice respectively lacking mPGES-1 and EP2receptors. RESULTS: IL-33 production by bmMFs requires endogenous PGE2 and intrinsic expression of EP2 receptor to amplify NF-kB-dependent LPSinduced IL-33 expression via exchange protein activated by cAMP (EPAC). Selective agonists of EP2 and to a lesser extent EP4, but not of EP1 or EP3, potentiated LPS-induced IL-33 generation from mPGES-1null and WT bmMFs. Exogenous PGE2 enhanced IL-33 mRNA expression via a cAMP dependent pathway and its effect was also mimicked by an EPAC-selective agonist, but not by a PKA-selective agonist, and was attenuated by an EPAC-selective antagonist or knockdown. Although both p38 MAPK and NF-kB activations were needed for IL-33 production, they occurred independently of PGE2. CONCLUSIONS: Our data demonstrate that endogenous PGE2 is required to amplify LPS-induced IL-33 expression in mouse bmMFs, involving cAMP dependent pathway involving EPAC. The ubiquitous induction of mPGES-1-dependent PGE2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders.
833
Environmental Adjuvants Induce Neuropilin-2 Expression in Human and Murine Alveolar Macrophages
Timothy P. Moran, MD, PhD1, Robert M. Immormino, PhD2, Hideki Nakano, PhD3, Neil Alexis, PhD4, Andrew J. Ghio, MD5, David B. Peden, MD, MS, FAAAAI6, and Donald N. Cook, PhD7; 1University of North Carolina School of Medicine, Chapel Hill, NC, 2Center for Environmental Medicine, Asthma and Lung Biology, Chapel Hill, NC, 3National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 4Center Environmental Med. Asthma/Lung Biology, Chapel Hill, NC, 5US EPA, Chapel Hill, NC, 6University of North Carolina at Chapel Hill School Medicine, Chapel Hill, NC, 7National Institute of Environmental Health Sciences, Research Triangle Park, NC. RATIONALE: Neuropilin-1 (NRP1) and -2 (NRP2) are transmembrane receptors expressed by lung macrophages, but their functional importance is unclear. Here, we investigated the effect of environmental adjuvants on NRP1 and NRP2 expression in human and murine alveolar macrophages (AM). METHODS: Human AM or murine bone marrow-derived macrophages (BMM) were treated ex vivo with Toll-like receptor (TLR) ligands, house dust extract (HDE) or proteases, and NRP1 and NRP2 expression was evaluated by real-time quantitative PCR and confocal microscopy. NRP2 reporter mice were also exposed to HDE or endotoxin (TLR4 agonist) in vivo, and NRP2 expression by AM was determined by flow cytometry. RESULTS: Ex vivo stimulation of human AM or murine BMM with endotoxin resulted in increased NRP2 but decreased NRP1 expression. NRP2 was also induced in BMM treated with HDE and agonists for TLR2,
TLR3 and TLR9. In contrast, protease treatment of BMM did not induce significant NRP2 expression. Expression of the NRP2 co-receptors, plexinA3 and vascular endothelial growth factor (VEGF) receptor 3, was increased in endotoxin- and HDE-stimulated BMM. In vivo airway exposure to HDE or endotoxin induced NRP2 expression in murine AM. Furthermore, quantitative PCR analysis revealed expression of NRP2 ligands (VEGF and semaphorin 3F) in lungs of HDE-exposed mice. CONCLUSIONS: Stimulation of innate immune receptors by TLR ligands and HDE induces NRP2 expression in human and murine AM. Given the importance of AM in maintaining immune homeostasis in the lungs, our findings suggest that NRP2 may play a role in regulating airway inflammation in response to environmental adjuvants.
834
Oxidative Burst Defines Differences in Respiratory Virus Associated Neutrophil Subtypes
Michelle Rohlfing1, Dorothy S. Cheung, MD, FAAAAI2, and Mitchell H. Grayson, MD, FAAAAI3; 1Nationwide Children’s Hospital, Columbus, OH, 2Genentech, South San Francisco, CA, 3700Children’s Drive, Nationwide Children’s Hospital / The Ohio State University, Columbus, OH. RATIONALE: Neutrophil (PMN) subsets are important in atopic disease development. In the Sendai virus (SeV) model of post-viral atopic disease, we demonstrated a critical need for CD49d+ PMN driving disease development. We showed humans express CD49d+ PMN and they track with atopy. However, unlike SeV, inoculation of mice with lipopolysaccharide (LPS) recruits CD49d– PMNs. Differences between CD49 expressing and nonexpressing PMNs are not well known. We undertook this study to determine if CD49d expression was associated with altered PMN oxidative burst. METHODS: C57BL/6 mice were inoculated intranasally with 2x105 pfu SeV. On day 3 post-inoculation, flow sorting obtained CD49d+ and CD49d– BAL PMN based on Gr-1/CD49d expression. Respiratory burst was examined using DHR assay (Cayman), with unstained subsets as controls. Additional PMN samples were cultured with 200 nM PMA for 45 min before DHR assay. BAL PMN (all CD49d–) from mice inoculated intranasally with 10 mg LPS one-day prior also were examined. RESULTS: SeV increased respiratory burst in CD49d+ compared to _4, CD49d– PMN (296630 Net MFI versus 114664, mean6sem, n> p50.03). PMA treatment had no effect (2856102 versus 7263, p50.05). However, LPS CD49d– PMN demonstrated significantly greater burst (312617 and 353625 PMA) than SeV CD49d– PMN (p50.03 for _0.3 for both). both), but not CD49d+ PMN (p> CONCLUSIONS: In SeV infection, CD49d+ have greater oxidative burst than CD49d– PMN; however, CD49d– PMN have an impaired burst compared to LPS CD49d– PMN. These data demonstrate diversity in PMN subtypes; further research will clarify the ‘‘PMN to atopy’’ axis.
MONDAY
832